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ABSTRACT Rhizobial attachment to host legume roots is the first physical interaction of bacteria and plants in symbiotic nitrogen fixation. The pH-dependent primary attachment of Rhizobium leguminosarum biovar viciae 3841 to Pisum sativum (pea) roots was investigated by genome-wide insertion sequencing, luminescence-based attachment assays, and proteomic analysis. Under acid, neutral, or alkaline pH, a total of 115 genes are needed for primary attachment under one or more environmental pH, with 22 genes required for all. These include components of cell surfaces and membranes, together with enzymes that construct and modify them. Mechanisms of dealing with stress also play a part; however, exact requirements vary depending on environmental pH. RNASeq showed that knocking out the two transcriptional regulators required for attachment causes massive changes in the bacterial cell surface. Approximately half of the 54 proteins required for attachment at pH 7.0 have a role in the later stages of nodule formation. We found no evidence for a single rhicadhesin responsible for alkaline attachment, although sonicated cell surface fractions inhibited root attachment at alkaline pH. Our results demonstrate the complexity of primary root attachment and illustrate the diversity of mechanisms involved.IMPORTANCEThe first step by which bacteria interact with plant roots is by attachment. In this study, we use a combination of insertion sequencing and biochemical analysis to determine how bacteria attach to pea roots and how this is influenced by pH. We identify several key adhesins, which are molecules that enable bacteria to stick to roots. This includes a novel filamentous hemagglutinin which is needed at all pHs for attachment. Overall, 115 proteins are required for attachment at one or more pHs.

ABSTRACT Biological nitrogen fixation in rhizobium-legume symbioses is of major importance for sustainable agricultural practices. To establish a mutualistic relationship with their plant host, rhizobia transition from free-living bacteria in soil to growth down infection threads inside plant roots and finally differentiate into nitrogen-fixing bacteroids. We reconstructed a genome-scale metabolic model for Rhizobium leguminosarum and integrated the model with transcriptome, proteome, metabolome, and gene essentiality data to investigate nutrient uptake and metabolic fluxes characteristic of these different lifestyles. Synthesis of leucine, polyphosphate, and AICAR is predicted to be important in the rhizosphere, while myo-inositol catabolism is active in undifferentiated nodule bacteria in agreement with experimental evidence. The model indicates that bacteroids utilize xylose and glycolate in addition to dicarboxylates, which could explain previously described gene expression patterns. Histidine is predicted to be actively synthesized in bacteroids, consistent with transcriptome and proteome data for several rhizobial species. These results provide the basis for targeted experimental investigation of metabolic processes specific to the different stages of the rhizobium-legume symbioses. IMPORTANCE Rhizobia are soil bacteria that induce nodule formation on plant roots and differentiate into nitrogen-fixing bacteroids. A detailed understanding of this complex symbiosis is essential for advancing ongoing efforts to engineer novel symbioses with cereal crops for sustainable agriculture. Here, we reconstruct and validate a genome-scale metabolic model for Rhizobium leguminosarum bv. viciae 3841. By integrating the model with various experimental data sets specific to different stages of symbiosis formation, we elucidate the metabolic characteristics of rhizosphere bacteria, undifferentiated bacteria inside root nodules, and nitrogen-fixing bacteroids. Our model predicts metabolic flux patterns for these three distinct lifestyles, thus providing a framework for the interpretation of genome-scale experimental data sets and identifying targets for future experimental studies.

ABSTRACT Pigeon pea, a legume crop native to India, is the primary source of protein for more than a billion people in developing countries. The plant can form symbioses with N2-fixing bacteria; however, reports of poor crop nodulation in agricultural soils abound. We report here a study of the bacterial community associated with pigeon pea, with a special focus on the symbiont population in different soils and vegetative and non-vegetative plant growth. Location with respect to the plant roots was determined to be the main factor controlling the bacterial community, followed by developmental stage and soil type. Plant genotype plays only a minor role. Pigeon pea roots have a reduced microbial diversity compared to the surrounding soil and select for Proteobacteria, especially for Rhizobium spp., during vegetative growth. While Bradyrhizobium, a native symbiont of pigeon pea, can be found associating with roots, its presence is dependent on plant variety and soil conditions. A combination of 16S rRNA gene amplicon survey, strain isolation, and co-inoculation with nodule-forming Bradyrhizobium spp. and non-N2-fixing Rhizobium spp. demonstrated that the latter is a much more successful colonizer of pigeon pea roots. Poor nodulation of pigeon pea in Indian soils may be caused by a poor Bradyrhizobium competitiveness against non-nodulating root colonizers such as Rhizobium. Hence, inoculant strain selection of symbionts for pigeon pea should be based not only on their nitrogen fixation potential but, more importantly, on their competitiveness in agricultural soils. IMPORTANCE Plant symbiosis with N2-fixing bacteria is a key to sustainable, low-input agriculture. While there are ongoing projects aiming to increase the yield of cereals using plant genetics and host-microbiota interaction engineering, the biggest potential lies in legume plants. Pigeon pea is a basic food source for a billion low-income people in India. Improving its interactions with N2-fixing rhizobia could dramatically reduce food poverty in India. Despite the Indian origin of this plant, pigeon pea nodulates only poorly in native soils. While there have been multiple attempts to select the best N2-fixing symbionts, there are no reliable strains available for geographically widespread use. In this article, using 16S rRNA gene amplicon, culturomics, and plant co-inoculation assays, we show that the native pigeon pea symbionts such as Bradyrhizobium spp. are able to nodulate their host, despite being poor competitors for colonizing roots. Hence, in this system, the establishment of effective symbiosis seems decoupled from microbial competition on plant roots. Thus, the effort of finding suitable symbionts should focus not only on their N2-fixing potential but also on their ability to colonize. Increasing pigeon pea yield is a low-hanging fruit to reduce world hunger and degradation of the environment through the overuse of synthetic fertilizers.

ABSTRACT Plant roots influence the soil microbiota via physical interaction, secretion, and plant immunity. However, it is unclear whether the root fraction or soil is more important in determining the structure of the prokaryotic or eukaryotic community and whether this varies between plant species. Furthermore, the leaf (phyllosphere) and root microbiotas have a large overlap; however, it is unclear whether this results from colonization of the phyllosphere by the root microbiota. Soil, rhizosphere, rhizoplane, and root endosphere prokaryote-, eukaryote-, and fungus-specific microbiotas of four plant species were analyzed with high-throughput sequencing. The strengths of factors controlling microbiota structure were determined using permutational multivariate analysis of variance (PERMANOVA) statistics. The origin of the phyllosphere microbiota was investigated using a soil swap experiment. Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root itself more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. A reciprocal soil swap experiment shows that the phyllosphere is colonized from the soil in which the plant is grown. IMPORTANCE Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root fraction more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined, indicating conserved adaptation of microbial communities to plants. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. We observed a remarkable similarity in the structure of a plant’s phyllosphere and root microbiotas and show by reciprocal soil swap experiments that both fractions are colonized from the soil in which the plant is grown. Thus, the phyllosphere is continuously colonized by the soil microbiota.

The purpose of this study was to define the pulmonary and systemic pharmacokinetics of colistin methanesulfonate (CMS) and formed colistin following intravenous (i.v.) and inhaled administration in cystic fibrosis (CF) patients. Six CF subjects were administered nebulized CMS doses of 2 and 4 million IU and an i.v. CMS infusion of 150 mg of colistin base activity. Blood plasma, sputum, and urine samples were collected for 12 to 24 h postdose. To assess the tolerability of the drug, lung function tests, blood serum creatinine concentrations, and adverse effect reports were recorded. All doses were well tolerated in the subjects. The pharmacokinetic parameters for CMS following i.v. delivery were consistent with previously reported values. Sputum concentrations of formed colistin were maintained at <1.0 mg/liter for 12 h postdose. Nebulization of CMS resulted in relatively high sputum concentrations of CMS and formed colistin compared to those resulting from i.v. administration. The systemic availability of CMS was low following nebulization of 2 and 4 million IU (7.93% ± 4.26% and 5.37% ± 1.36%, respectively), and the plasma colistin concentrations were below the limit of quantification. Less than 2 to 3% of the nebulized CMS dose was recovered in the urine samples in 24 h. The therapeutic availability and drug targeting index for CMS and colistin following inhalation compared to i.v. delivery were significantly greater than 1. Inhalation of CMS is an effective means of targeting CMS and formed colistin for delivery to the lungs, as high lung exposure and minimal systemic exposure were achieved in CF subjects.

Much attention has focused on the Mycobacterium tuberculosis molecular chaperone chaperonin (Cpn) 60.2 (Hsp 65) in the pathology of tuberculosis because of its immunogenicity and ability to directly activate human monocytes and vascular endothelial cells. However, M. tuberculosis is one of a small group of bacteria that contain multiple genes encoding Cpn 60 proteins. We have now cloned and expressed both M. tuberculosis proteins and report that the novel chaperonin 60, Cpn 60.1, is a more potent inducer of cytokine synthesis than is Cpn 60.2. This is in spite of 76% amino acid sequence similarity between the two mycobacterial chaperonins. The M. tuberculosis Cpn 60.2 protein activates human peripheral blood mononuclear cells by a CD14-independent mechanism, whereas Cpn 60.1 is partially CD14 dependent and contains a peptide sequence whose actions are blocked by anti-CD14 monoclonal antibodies. The cytokine-inducing activity of both chaperonins is extremely resistant to heat. Cpn 60.1 may be an important virulence factor in tuberculosis, able to activate cells by diverse receptor-driven mechanisms.

It has recently been discovered that Actinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC. In this study, we have cloned and expressed each toxin gene from A. actinomycetemcomitans in Escherichia coli and purified the recombinant Cdt proteins to homogeneity. Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial cell line HEp-2. The only combinations of toxin proteins causing cell cycle arrest were the presence of all three Cdt proteins and the combination of CdtB and CdtC. A similar experimental protocol was used to determine if recombinant Cdt proteins were able to induce human peripheral blood mononuclear cells (PBMCs) to produce cytokines. The individual Cdt proteins were able to induce the synthesis by PBMCs of interleukin-1beta (IL-1beta), IL-6, and IL-8 but not of tumor necrosis factor alpha, IL-12, or granulocyte-macrophage colony-stimulating factor, with CdtC being the most potent and CdtB being the least potent cytokine inducer. There was evidence of synergism between these Cdt proteins in the stimulation of cytokine production, most markedly with gamma interferon, which required the minimum interaction of CdtB and -C to stimulate production.

A phoA fusion library of Actinobacillus actinomycetemcomitans genomic DNA has been screened to identify genes encoding exported and secreted proteins. A total of 8,000 colonies were screened, and 80 positive colonies were detected. From these, 48 genes were identified with (i) more than half having homology to known or hypothetical Haemophilus influenzae genes, (ii) 14 having no ascribed function, and (iii) 4 having very limited or no homology to known genes. The proteins encoded by these genes may, by virtue of their presence on the cell surface, be novel virulence determinants.

Thioredoxin is a ubiquitous redox control and cell stress protein. Unexpectedly, in recent years, thioredoxins have been found to exhibit both cytokine and chemokine activities, and there is increasing evidence that this class of protein plays a role in the pathogenesis of inflammatory diseases. In spite of this evidence, it has been reported that the oral bacterium and periodontopathogen Actinobacillus actinomycetemcomitans secretes an immunosuppressive factor (termed suppressive factor 1 [SF1] [T. Kurita-Ochiai and K. Ochiai, Infect. Immun. 64:50-54, 1996]) whose N-terminal sequence, we have determined, identifies it as thioredoxin. We have cloned and expressed the gene encoding the thioredoxin of A. actinomycetemcomitans and have purified the protein to homogeneity. The A. actinomycetemcomitans trx gene has 52 and 76% identities, respectively, to the trx genes of Escherichia coli and Haemophilus influenzae. Enzymatic analysis revealed that the recombinant protein had the expected redox activity. When the recombinant thioredoxin was tested for its capacity to inhibit the production of cytokines by human peripheral blood mononuclear cells, it showed no significant inhibitory capacity. We therefore conclude that the thioredoxin of A. actinomycetemcomitans does not act as an immunosuppressive factor, at least with human leukocytes in cultures, and that the identity of SF1 remains to be elucidated.

We report the discovery of a novel genetic locus within Staphylococcus aureus that encodes a cluster of at least five exotoxin-like proteins. Designated the staphylococcal exotoxin-like genes 1 to 5 (set1 to set5), these open reading frames have between 38 and 53% homology to each other. All five proteins contain consensus sequences that are found in staphylococcal and streptococcal exotoxins and toxic shock syndrome toxin 1 (TSST-1). However, the SETs have only limited overall sequence homology to the enterotoxins and TSST-1 and thus represent a novel family of exotoxin-like proteins. The prototypic gene in this cluster, set1, has been cloned and expressed. Recombinant SET1 stimulated the production of interleukin-1beta, interleukin-6, and tumor necrosis factor alpha by human peripheral blood mononuclear cells. PCR analysis revealed that set1 was distributed among other strains of S. aureus but not in the other staphylococcal species examined. Sequence analysis of the set1 genes from different strains revealed at least three allelic variants. The protein products of these allelic variants displayed a 100-fold difference in their cytokine-inducing potency. The distribution of allelic variants of the set genes among strains of S. aureus may contribute to differences in the pathogenic potential of this bacterium.

Cytokines are a diverse group of proteins and glycoproteins which have potent and wide-ranging effects on eukaryotic cell function and are now recognized as important mediators of tissue pathology in infectious diseases. It is increasingly recognized that for many bacterial species, cytokine induction is a major virulence mechanism. Until recent years, the only bacterial component known to stimulate cytokine synthesis was lipopolysaccharide (LPS). It is only within the past decade that it has been clearly shown that many components associated with the bacterial cell wall, including proteins, glycoproteins, lipoproteins, carbohydrates, and lipids, have the capacity to stimulate mammalian cells to produce a diverse array of cytokines. It has been established that many of these cytokine-inducing molecules act by mechanisms distinct from that of LPS, and thus their activities are not due to LPS contamination. Bacteria produce a wide range of virulence factors which cause host tissue pathology, and these diverse factors have been grouped into four families: adhesins, aggressins, impedins, and invasins. We suggest that the array of bacterial cytokine-inducing molecules represents a new class of bacterial virulence factor, and, by analogy with the known virulence families, we suggest the term "modulin" to describe these molecules, because the action of cytokines is to modulate eukaryotic cell behavior. This review summarizes our current understanding of cytokine biology in relation to tissue homeostasis and disease and concisely reviews the current literature on the cytokine-inducing molecules produced by gram-negative and gram-positive bacteria, with an emphasis on the cellular mechanisms responsible for cytokine induction. We propose that modulins, by controlling the host immune and inflammatory responses, maintain the large commensal flora that all multicellular organisms support.